技术资料

This protocol describes an EDTA-based passaging procedure to be used with chemically defined E8 medium that serves as a tool for basic and translational research into human pluripotent stem cells (PSCs). In this protocol,passaging one six-well or 10-cm plate of cells takes about 6-7 min. This enzyme-free protocol achieves maximum cell survival without enzyme neutralization,centrifugation or drug treatment. It also allows for higher throughput,requires minimal material and limits contamination. Here we describe how to produce a consistent E8 medium for routine maintenance and reprogramming and how to incorporate the EDTA-based passaging procedure into human induced PSC (iPSC) derivation,colony expansion,cryopreservation and teratoma formation. This protocol has been successful in routine cell expansion,and efficient for expanding large-volume cultures or a large number of cells with preferential dissociation of PSCs. Effective for all culture stages,this procedure provides a consistent and universal approach to passaging human PSCs in E8 medium. View Publication

Recent evidence suggests human embryonic stem cell (hESC) and induced pluripotent stem (iPS) cell lines have differences in their epigenetic marks and transcriptomes,yet the impact of these differences on subsequent terminally differentiated cells is less well understood. Comparison of purified,homogeneous populations of somatic cells derived from multiple independent human iPS and ES lines will be required to address this critical question. Here,we report a differentiation protocol based on embryonic development that consistently yields large numbers of endothelial cells (ECs) derived from multiple hESCs or iPS cells. Mesoderm differentiation of embryoid bodies was maximized,and defined growth factors were used to generate KDR+ EC progenitors. Magnetic purification of a KDR+ progenitor subpopulation resulted in an expanding,homogeneous pool of ECs that expressed EC markers and had functional properties of ECs. Comparison of the transcriptomes revealed limited gene expression variability between multiple lines of human iPS-derived ECs or between lines of ES- and iPS-derived ECs. These results demonstrate a method to generate large numbers of pure human EC progenitors and differentiated ECs from pluripotent stem cells and suggest individual lineages derived from human iPS cells may have significantly less variance than their pluripotent founders. STEM Cells2013;31:92–103 View Publication

Trisomy 21 is associated with hematopoietic abnormalities in the fetal liver,a preleukemic condition termed transient myeloproliferative disorder,and increased incidence of acute megakaryoblastic leukemia. Human trisomy 21 pluripotent cells of various origins,human embryonic stem (hES),and induced pluripotent stem (iPS) cells,were differentiated in vitro as a model to recapitulate the effects of trisomy on hematopoiesis. To mitigate clonal variation,we isolated disomic and trisomic subclones from the same parental iPS line,thereby generating subclones isogenic except for chromosome 21. Under differentiation conditions favoring development of fetal liver-like,γ-globin expressing,definitive hematopoiesis,we found that trisomic cells of hES,iPS,or isogenic origins exhibited a two- to fivefold increase in a population of CD43(+)(Leukosialin)/CD235(+)(Glycophorin A) hematopoietic cells,accompanied by increased multilineage colony-forming potential in colony-forming assays. These findings establish an intrinsic disturbance of multilineage myeloid hematopoiesis in trisomy 21 at the fetal liver stage. View Publication

BACKGROUND: The histone variant H2A.Z has been implicated in nucleosome exchange,transcriptional activation and Polycomb repression. However,the relationships among these seemingly disparate functions remain obscure.backslashnbackslashnRESULTS: We mapped H2A.Z genome-wide in mammalian ES cells and neural progenitors. H2A.Z is deposited promiscuously at promoters and enhancers,and correlates strongly with H3K4 methylation. Accordingly,H2A.Z is present at poised promoters with bivalent chromatin and at active promoters with H3K4 methylation,but is absent from stably repressed promoters that are specifically enriched for H3K27 trimethylation. We also characterized post-translational modification states of H2A.Z,including a novel species dually-modified by ubiquitination and acetylation that is enriched at bivalent chromatin.backslashnbackslashnCONCLUSIONS: Our findings associate H2A.Z with functionally distinct genomic elements,and suggest that post-translational modifications may reconcile its contrasting locations and roles. View Publication

FUS/TLS (fused in sarcoma/translocated in liposarcoma) and TDP-43 are integrally involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We found that FUS/TLS binds to RNAs from textgreater5,500 genes in mouse and human brain,primarily through a GUGGU-binding motif. We identified a sawtooth-like binding pattern,consistent with co-transcriptional deposition of FUS/TLS. Depletion of FUS/TLS from the adult nervous system altered the levels or splicing of textgreater950 mRNAs,most of which are distinct from RNAs dependent on TDP-43. Abundance of only 45 RNAs was reduced after depletion of either TDP-43 or FUS/TLS from mouse brain,but among these were mRNAs that were transcribed from genes with exceptionally long introns and that encode proteins that are essential for neuronal integrity. Expression levels of a subset of these were lowered after TDP-43 or FUS/TLS depletion in stem cell-derived human neurons and in View Publication

Immature primary and stem cell-derived cardiomyocytes provide useful models for fundamental studies of heart development and cardiac disease,and offer potentialbackslashrbackslashnfor patient specific drug testing and differentiation protocols aimed at cardiac grafts. To assess their potential for augmenting heart function,and to gain insight into cardiac growth and disease,tissue engineers must quantify the contractile forces of these single cells. Currently,axial contractile forces of isolated adult heart cells can only be measuredbackslashrbackslashnby two-point methods such as carbon fiber techniques,which cannot be applied to neonatal and stem cell-derived heart cells because they are more difficult to handle and lack a persistent shape. Here we present a novel axial technique for measuring the contractile forces of isolated immature cardiomyocytes. We overcome cell manipulation and patterning challenges by using a thermoresponsive sacrificialbackslashrbackslashnsupport layer in conjunction with arrays of widely separated elastomeric microposts. Our approach has the potential to be high-throughput,is functionally analogous to current gold-standard axial force assays for adult heart cells,and prescribes elongated cell shapes without protein patterning. Finally,we calibrate these force posts withbackslashrbackslashnpiezoresistive cantilevers to dramatically reduce measurement error typical for soft polymer-based force assays. We report quantitative measurements of peak contractile forces up to 146 nN with post stiffness standard error (26 nN) far betterbackslashrbackslashnthan that based on geometry and stiffness estimates alone. The addition of sacrificial layers to future 2D and 3D cell culturebackslashrbackslashnplatforms will enable improved cell placement and the complex suspension of cells across 3D constructs. View Publication

Cell Research 23,122 (2013). doi:10.1038/cr.2012.119 View Publication

The Hippo pathway is crucial in organ size control,and its dysregulation contributes to tumorigenesis. However,upstream signals that regulate the mammalian Hippo pathway have remained elusive. Here,we report that the Hippo pathway is regulated by G-protein-coupled receptor (GPCR) signaling. Serum-borne lysophosphatidic acid (LPA) and sphingosine 1-phosphophate (S1P) act through G12/13-coupled receptors to inhibit the Hippo pathway kinases Lats1/2,thereby activating YAP and TAZ transcription coactivators,which are oncoproteins repressed by Lats1/2. YAP and TAZ are involved in LPA-induced gene expression,cell migration,and proliferation. In contrast,stimulation of Gs-coupled receptors by glucagon or epinephrine activates Lats1/2 kinase activity,thereby inhibiting YAP function. Thus,GPCR signaling can either activate or inhibit the Hippo-YAP pathway depending on the coupled G protein. Our study identifies extracellular diffusible signals that modulate the Hippo pathway and also establishes the Hippo-YAP pathway as a critical signaling branch downstream of GPCR. View Publication

Although we previously reported the development of cell-dense thickened cardiac tissue by repeated transplantation-based vascularization of neonatal rat cardiac cell sheets,the cell sources for human cardiac cells sheets and their functions have not been fully elucidated. In this study,we developed a bioreactor to expand and induce cardiac differentiation of human induced pluripotent stem cells (hiPSCs). Bioreactor culture for 14 days produced around 8×10(7) cells/100 ml vessel and about 80% of cells were positive for cardiac troponin T. After cardiac differentiation,cardiomyocytes were cultured on temperature-responsive culture dishes and showed spontaneous and synchronous beating,even after cell sheets were detached from culture dishes. Furthermore,extracellular action potential propagation was observed between cell sheets when two cardiac cell sheets were partially overlaid. These findings suggest that cardiac cell sheets formed by hiPSC-derived cardiomyocytes might have sufficient properties for the creation of thickened cardiac tissue. View Publication

Human embryonic stem cells (hESCs) are a promising cell source for tissue engineering and regenerative medicine,but before they can be used in therapies,we must be able to accurately identify the state and progeny of hESCs. One of the most commonly used methods for identification is flow cytometry. Many flow cytometry applications use antibodies to detect the amount of antigen present on/in a cell. This allows for the identification of unique cell populations or the tracking of expression changes within a population during differentiation. The results are typically presented as a percentage of positively expressing cells (%Pos) for a marker of choice,relative to a negative control. However,this reporting term is vulnerable to distortion from outliers and inaccuracy from loss of information about the population's fluorescence intensity. In this article,we describe an alternate strategy that uses the normalized median fluorescence intensity (nMFI),in which the MFI of the stained sample is normalized to the MFI of the negative control,as the reporting term to more accurately describe a population of cells in culture. We observed that nMFI provides a more accurate representation for the quality of a starting population and comparing data of different experimental runs. In addition,we demonstrated that the nMFI is a more sensitive measure of pluripotent and differentiation markers expression changes during hESC differentiation into three germ layer lineages. View Publication

Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSCs) can be differentiated to neural cells that model neurodegenerative diseases and be used in the screening of potential drugs to ameliorate the disease phenotype. Traditionally,NPCs are produced in 2D cultures,in low yields,using a laborious process that includes generation of embryonic bodies,plating,and colony selections. To simplify the process and generate large numbers of hiPSC-derived NPCs,we introduce a microcarrier (MC) system for the expansion of a hiPSC line and its subsequent differentiation to NPC,using iPS (IMR90) as a model cell line. In the expansion stage,a process of cell propagation in serum-free MC culture was developed first in static culture,which is then scaled up in stirred spinner flasks. A 7.7-fold expansion of iPS (IMR90) and cell yield of 1.3×10�?� cells/mL in 7 days of static MC culture were achieved. These cells maintained expression of OCT 3/4 and TRA-1-60 and possessed a normal karyotype over 10 passages. A higher cell yield of 6.1×10�?� cells/mL and 20-fold hiPSC expansion were attained using stirred spinner flasks (seeded from MC static cultures) and changing the medium-exchange regimen from once to twice a day. In the differentiation stage,NPCs were generated with 78%-85% efficiency from hiPSCs using a simple serum-free differentiation protocol. Finally,the integrated process of cell expansion and differentiation of hiPSCs into NPCs using an MC in spinner flasks yielded 333 NPCs per seeded hiPSC as compared to 53 in the classical 2D tissue culture protocol. Similar results were obtained with the HES-3 human embryonic stem cell line. These NPCs were further differentiated into βIII-tubulin�?� neurons,GFAP�?� astrocytes,and O4�?� oligodendrocytes,showing that cells maintained their multilineage differentiation potential. View Publication

Human embryonic stem cells (hESCs) are self-renewing,pluripotent cells derived from the inner cell mass of blastocysts,early-stage embryos,or blastomeres. hESCs can be propagated indefinitely in an undifferentiated state in vitro and have the ability to differentiate into all cell types of the body. Therefore,these cells can potentially provide an unlimited source of cells and hold promise for transplantation therapy,regenerative medicine,drug screening and discovery,and basic scientific research. Surplus human embryos donated for hESC derivation are extremely valuable,and inefficient derivation of hESCs would be a terrible waste of human embryos. Here,we describe a method for isolating hESC lines from human blastocysts with high efficiency. We also describe the methods for excising differentiated areas from partially differentiated hESC colonies and re-isolating undifferentiated hESCs from extremely differentiated hESC colonies. View Publication